The unfolded protein response (UPR) is a signal transduction pathway that allows cells to survive environmental stresses that perturb protein folding and maturation in the endoplasmic reticulum (ER) (Ma and Hendershot, 2004), (Feldman et al., 2005), (Koumenis and Wouters, 2006). Stress stimuli that activate UPR include hypoxia, disruption of protein glycosylation (glucose deprivation), depletion of luminal ER calcium, or changes in ER redox status (Ma and Hendershot, 2004), (Feldman et al., 2005). These perturbations result in the accumulation of unfolded or mis-folded proteins in the ER, which is sensed by resident ER membrane proteins. These proteins activate a coordinated cellular response to alleviate the impact of the stress and enhance cell survival. Responses include an increase in the level of chaperone proteins to enhance protein re-folding, degradation of the mis-folded proteins, and translational arrest to decrease the burden of proteins entering the ER. These pathways also regulate cell survival by modulating apoptosis (Ma and Hendershot, 2004), (Feldman et al., 2005), (Hamanaka et al., 2009) and autophagy (Rouschop et al.), and can trigger cell death under conditions of prolonged ER stress.
Three ER membrane proteins have been identified as primary effectors of the UPR: protein kinase R (PKR)-like ER kinase [PERK, also known as eukaryotic initiation factor 2A kinase 3 (EIF2AK3), or pancreatic eIF2α kinase (PEK)], inositol-requiring gene 1 α/β (IRE1), and activating transcription factor 6 (ATF6) (Ma and Hendershot, 2004). Under normal conditions these proteins are held in the inactive state by binding to the ER chaperone, GRP78 (BiP). Accumulation of unfolded proteins in the ER leads to release of GRP78 from these sensors resulting in their activation (Ma et al., 2002). PERK is a type I ER membrane protein containing a stress-sensing domain facing the ER lumen, a transmembrane segment, and a cytosolic kinase domain (Shi et al., 1998), (Sood et al., 2000). Release of GRP78 from the stress-sensing domain of PERK results in oligomerization and autophosphorylation at multiple serine, threonine and tyrosine residues (Ma et al., 2001), (Su et al., 2008). The major substrate for PERK is the eukaryotic initiation factor 2α (eIF2α) at serine-51 (Marciniak et al., 2006). This site is also phosphorylated by other PERK family members [(general control non-derepressed 2 (GCN2), PKR, and heme-regulated kinase (HRI)] in response to different stimuli, and by pharmacological inducers of ER stress such as thapsigargin and tunicamycin. Phosphorylation of eIF2α converts it to an inhibitor of eIF2B, which hinders the assembly of the 40S ribosome translation initiation complex and consequently reduces the rate of translation initiation. Among other effects, this leads to a loss of cyclin D1 in cells resulting in arrest in the G1 phase of the cell division cycle (Brewer and Diehl, 2000), (Hamanaka et al., 2005). Paradoxically, translation of certain messages encoding downstream effectors of eIF2α, ATF4 and CHOP (C/EBP homologous protein; GADD153), which modulate cellular survival pathways, is actually increased upon ER stress. A second PERK substrate, Nrf2, regulates cellular redox potential, contributes to cell adaptation to ER stress, and promotes survival (Cullinan and Diehl, 2004). The normal function of PERK is to protect secretory cells from ER stress. Phenotypes of PERK knockout mice include diabetes, due to loss of pancreatic islet cells, skeletal abnormalities, and growth retardation (Harding et al., 2001), (Zhang et al., 2006), (Iida et al., 2007). These features are similar to those seen in patients with Wolcott-Rallison syndrome, who carry germline mutations in the PERK gene (Delepine et al., 2000). IRE1 is a transmembrane protein with kinase and endonulease (RNAse) functions (Feldman et al., 2005) (Koumenis and Wouters, 2006). Under ER stress, it undergoes oligomerization and autophosphorylation, which activates the endonuclease to excise an intron from unspliced X-box binding protein 1 (XBP1) mRNA. This leads to the synthesis of truncated XBP1s, which activates transcription of UPR genes. The third effector of UPR, ATF6, is transported to the golgi upon ER stress, where it is cleaved by proteases to release the cytosolic transcription domain. This domain translocates to the nucleus and activates transcription of UPR genes (Feldman et al., 2005), (Koumenis and Wouters, 2006).
Tumor cells experience episodes of hypoxia and nutrient deprivation during their growth due to inadequate blood supply and aberrant blood vessel function (Brown and Wilson, 2004), (Blais and Bell, 2006). Thus, they are likely to be dependent on active UPR signaling to facilitate their growth. Consistent with this, mouse fibroblasts derived from PERK−/−, XBP1−/−, and ATF4−/− mice, and fibroblasts expressing mutant eIF2α show reduced clonogenic growth and increased apoptosis under hypoxic conditions in vitro and grow at substantially reduced rates when implanted as tumors in nude mice (Koumenis et al., 2002), (Romero-Ramirez et al., 2004), (Bi et al., 2005). Human tumor cell lines carrying a dominant negative PERK that lacks kinase activity also showed increased apoptosis in vitro under hypoxia and impaired tumor growth in vivo (Bi et al., 2005). In these studies, activation of the UPR was observed in regions within the tumor that coincided with hypoxic areas. These areas exhibited higher rates of apoptosis compared to tumors with intact UPR signaling. Further evidence supporting the role of PERK in promoting tumor growth is the observation that the number, size, and vascularity of insulinomas arising in transgenic mice expressing the SV40-T antigen in the insulin-secreting beta cells, was profoundly reduced in PERK −/− mice compared to wild-type control (Gupta et al., 2009). Activation of the UPR has also been observed in clinical specimens. Human tumors, including those derived from cervical carcinomas, glioblastomas (Bi et al., 2005), lung cancers (Jorgensen et al., 2008) and breast cancers (Ameri et al., 2004), (Davies et al., 2008) show elevated levels of proteins involved in UPR, compared to normal tissues. Therefore, inhibiting the unfolded protein response with compounds that block the activity of PERK and other components of the UPR is expected to have utility as anticancer agents and in the treatment of diseases associated with activated unfolded protein response pathways, such as Alzheimer's disease, stroke and Type 1 diabetes.
Loss of endoplasmic reticulum homeostasis and accumulation of misfolded proteins can contribute to a number of disease states including cardiovascular and degenerative diseases (Paschen, 2004) such as: Alzheimer's disease (Salminen et al., 2009 and O'Connor et. al. 2008), Parkinson disease, Huntington's disease, amyotrophic lateral sclerosis (Kanekura et. al., 2009 and Nassif et. al. 2010), myocardial infarction, cardiovascular disease, atherosclerosis (McAlpine et. al, 2010), and arrhythmias. A PERK inhibitor is expected to have utility in the treatment of such cardiovascular and degenerative diseases in which the underlying pathology and symptoms are associated with dysregulaton of the unfolded protein response.